Single-stranded oligodeoxynucleotides induce plant defence in Arabidopsis thaliana

Background and AimsSingle-stranded DNA oligodeoxynucleotides (ssODNs) have been shown to elicit immune responses in mammals. In plants, RNA and genomic DNA can activate immunity, although the exact mechanism through which they are sensed is not clear. The aim of this work was to study the possible effect of ssODNs on plant immunity.Key ResultsThe ssODNs IMT504 and 2006 increased protection against the pathogens Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea but not against tobacco mosaic virus-Cg when infiltrated in Arabidopsis thaliana. In addition, ssODNs inhibited root growth and promoted stomatal closure in a concentration-dependent manner, with half-maximal effective concentrations between 0.79 and 2.06 µM. Promotion of stomatal closure by ssODNs was reduced by DNase I treatment. It was also diminished by the NADPH oxidase inhibitor diphenyleneiodonium and by coronatine, a bacterial toxin that inhibits NADPH oxidase-dependent reactive oxygen species (ROS) synthesis in guard cells. In addition it was found that ssODN-mediated stomatal closure was impaired in bak1-5, bak1-5/bkk1, mpk3 and npr1-3 mutants. ssODNs also induced early expression of MPK3, WRKY33, PROPEP1 and FRK1 genes involved in plant defence, an effect that was reduced in bak1-5 and bak1-5/bkk1 mutants.ConclusionsssODNs are capable of inducing protection against pathogens through the activation of defence genes and promotion of stomatal closure through a mechanism similar to that of other elicitors of plant immunity, which involves the BAK1 co-receptor, and ROS synthesis.Fil: Toum, Laila. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Conti, Gabriela. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Coppola Guerriero, Francesca Marina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; ArgentinaFil: Conforte, Valeria Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Garolla, Franco A.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Biodiversidad y Biología Experimental; ArgentinaFil: Asurmendi, Sebastian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación En Ciencias Veterinarias y Agronómicas. Instituto de Agrobiotecnología y Biología Molecular. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Agrobiotecnología y Biología Molecular; ArgentinaFil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Gudesblat, Gustavo Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Biodiversidad y Biología Experimental y Aplicada. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biociencias, Biotecnología y Biología Traslacional.; Argentin

Controlled synthesis of the DSF cell–cell signal is required for biofilm formation and virulence in Xanthomonas campestris

Virulence of the black rot pathogen Xanthomonas campestris pv. campestris (Xcc) is regulated by cell–cell signalling involving the diffusible signal factor DSF. Synthesis and perception of DSF require products of genes within the rpf cluster (for regulation of pathogenicity factors). RpfF directs DSF synthesis whereas RpfC and RpfG are involved in DSF perception. Here we have examined the role of the rpf/DSF system in biofilm formation in minimal medium using confocal laser-scanning microscopy of GFP-labelled bacteria. Wild-type Xcc formed microcolonies that developed into a structured biofilm. In contrast, an rpfF mutant (DSF-minus) and an rpfC mutant (DSF overproducer) formed only unstructured arrangements of bacteria. A gumB mutant, defective in xanthan biosynthesis, was also unable to develop the typical wild-type biofilm. Mixed cultures of gumB and rpfF mutants formed a typical biofilm in vitro. In contrast, in mixed cultures the rpfC mutant prevented the formation of the structured biofilm by the wild-type and did not restore wild-type biofilm phenotypes to gumB or rpfF mutants. These effects on structured biofilm formation were correlated with growth and disease development by Xcc strains in Nicotiana benthamiana leaves. These findings suggest that DSF signalling is finely balanced during both biofilm formation and virulence

Structural basis for the Pr-Pfr long-range signaling mechanism of a full-length bacterial phytochrome at the atomic level

Phytochromes constitute a widespread photoreceptor family that typically interconverts between two photostates called Pr (red light–absorbing) and Pfr (far-red light–absorbing). The lack of full-length structures solved at the (near-)atomic level in both pure Pr and Pfr states leaves gaps in the structural mechanisms involved in the signal transmission pathways during the photoconversion. Here, we present the crystallographic structures of three versions from the plant pathogen Xanthomonas campestris virulence regulator XccBphP bacteriophytochrome, including two full-length proteins, in the Pr and Pfr states. The structures show a reorganization of the interaction networks within and around the chromophore-binding pocket, an α-helix/β-sheet tongue transition, and specific domain reorientations, along with interchanging kinks and breaks at the helical spine as a result of the photoswitching, which subsequently affect the quaternary assembly. These structural findings, combined with multidisciplinary studies, allow us to describe the signaling mechanism of a full-length bacterial phytochrome at the atomic level.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikEC/H2020/664726/EU/EMBL Interdisciplinary, International and Intersectorial Postdocs/EI3PO

Xanthomonas campestris Overcomes Arabidopsis Stomatal Innate Immunity through a DSF Cell-to-Cell Signal-Regulated Virulence Factor1[OA]

Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host. In this article, we describe a factor secreted by the bacterial phytopathogen Xanthomonas campestris pv campestris (Xcc) capable of interfering with stomatal closure induced by bacteria or abscisic acid (ABA). We found that living Xcc, as well as ethyl acetate extracts from Xcc culture supernatants, are capable of reverting stomatal closure induced by bacteria, lipopolysaccharide, or ABA. Xcc ethyl acetate extracts also complemented the infectivity of Pseudomonas syringae pv tomato (Pst) mutants deficient in the production of the coronatine toxin, which is required to overcome stomatal defense. By contrast, the rpfF and rpfC mutant strains of Xcc, which are unable to respectively synthesize or perceive a diffusible molecule involved in bacterial cell-to-cell signaling, were incapable of reverting stomatal closure, indicating that suppression of stomatal response by Xcc requires an intact rpf/diffusible signal factor system. In addition, we found that guard cell-specific Arabidopsis (Arabidopsis thaliana) Mitogen-Activated Protein Kinase3 (MPK3) antisense mutants were unresponsive to bacteria or lipopolysaccharide in promotion of stomatal closure, and also more sensitive to Pst coronatine-deficient mutants, showing that MPK3 is required for stomatal immune response. Additionally, we found that, unlike in wild-type Arabidopsis, ABA-induced stomatal closure in MPK3 antisense mutants is not affected by Xcc or by extracts from Xcc culture supernatants, suggesting that the Xcc factor might target some signaling component in the same pathway as MPK3

Micro ARNs involucrados en la respuesta a Xanthomonas SPP, en Citrus Limon

Poster presented at the VIII Encuentro para Latino América y el Caribe de Biotecnología REDBIO Argentina 2013, held in Mar de Plata on November 18-22, 2013. More details at http://redbioargentina2013.com.ar/.Las plantas responden a la infección por patógenos a través de un sistema inmune innato basado en el reconocimiento de moléculas asociadas al patógeno o PAMPs. Esta respuesta se denomina inmunidad inducida por PAMPs o PTI (por, PAMP-triggered immunity). Algunos PAMPs inducen la expresión de determinados micro ARNs (miR), los cuales regulan la transducción de señales hormonales y la expresión de genes de defensa, inhibiendo la colonización del patógeno. Sin embargo, los patógenos exitosos han desarrollando estrategias altamente especializadas para suprimir PTI, conduciendo de este modo a la enfermedad. Xanthomonas citri subsp. citri (X. citri) es la bacteria causal del cancro cítrico. Todos los cultivares de cítricos son susceptibles a X. citri.Peer reviewe

Xanthomonas vesicatoria virulence factors involved in early stages of bacterial spot development in tomato

Xanthomonas vesicatoria (Xv) is a member of a species complex that causes bacterial spot on tomato, one of the most important diseases of this crop worldwide. The objective of this investigation was to analyse several characteristics involved in Xv virulence in relation to strain aggressiveness. Motility, biofilm formation, adhesion and production of xanthan were evaluated in three local strains causing tomato bacterial spot in Argentina. The strains assayed presented differential swarming and twitching motilities, adhesion and biofilm formation abilities. The most aggressive strain, BNM 208, exhibited the greatest swarming and twitching motilities, and developed a mature biofilm with presence of defined cell clusters, a homogeneous and compact structure, and higher biomass and substratum coverage than the other two strains. Even though the three strains produced similar amounts of xanthan, BNM 208 produced the most viscous exopolysaccharide, which possibly relates to the better characteristics of its biofilm. Despite other differences, the three strains multiplied to similar levels when they were infiltrated into the leaf. The results suggest that the aggressiveness of Xv strains studied in this work was related to their ability to move by flagella or type IV pili, adhere to leaves and form well developed biofilms, factors that improve phyllosphere colonization. A better understanding of the factors involved in the Xv infection process at the early stages would contribute to developing new control strategies for this phytopathogen.Fil: Felipe, Verónica. Universidad Nacional de Villa María; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romero, A. M.. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Producción Vegetal; ArgentinaFil: Montecchia, Marcela Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; ArgentinaFil: Vojnov, Adrián Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Bianco, María Isabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Ciencia y Tecnología "Dr. César Milstein". Fundación Pablo Cassará. Instituto de Ciencia y Tecnología "Dr. César Milstein"; ArgentinaFil: Yaryura, Pablo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Centro de Investigaciones y Transferencia de Villa María. Universidad Nacional de Villa María. Centro de Investigaciones y Transferencia de Villa María; Argentin

The histone-like protein HupB influences biofilm formation and virulence in Xanthomonas citri ssp. citri through the regulation of flagellar biosynthesis

Citrus canker is an important disease of citrus, whose causal agent is the bacterium Xanthomonas citri ssp. citri (Xcc). In previous studies, we found a group of Xcc mutants, generated by the insertion of the Tn5 transposon, which showed impaired ability to attach to an abiotic substrate. One of these mutants carries the Tn5 insertion in hupB, a gene encoding a bacterial histone-like protein, homologue to the β-subunit of the Heat-Unstable (HU) nucleoid protein of Escherichia coli. These types of protein are necessary to maintain the bacterial nucleoid organization and the global regulation of gene expression. Here, we characterized the influence of the mutation in hupB regarding Xcc biofilm forma tion and virulence. The mutant strain hupB was incapable of swimming in soft agar, whereas its complemented strain partially recovered this phenotype. Electron microscope imaging revealed that impaired motility of hupB was a consequence of the absence of the flagellum. Comparison of the expression of flagellar genes between the wild-type strain and hupB showed that the mutant exhibited decreased expression of fliC (encoding flagellin). The hupB mutant also displayed reduced virulence compared with the wild-type strain when they were used to infect Citrus lemon plants using different infection methods. Our results therefore show that the histone-like protein HupB plays an essential role in the pathogenesis of Xcc through the regulation of biofilm formation and biosynthesis of the flagellum.Fil: Conforte, Valeria P. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: Malamud, Florencia. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina.Fil: Yaryura, Pablo M. Universidad de Villa María. Centro de Investigaciones y Transferencia de Villa María (CIT - CONICET); Argentina.Fil: Toum Terrones, Laila. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina.Fil: Toum Terrones, Laila. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA - CONICET); Argentina.Fil: Torres, Pablo S. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: De Pino, Verónica. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: Chazarreta, Cristian N. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina.Fil: Gudesblat, Gustavo E. Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA – CONICET -Estación Experimental Agroindustrial Obispo Colombres (EEAOC); Argentina.Fil: Castagnaro, Atilio P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina.Fil: Castagnaro, Atilio P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Biodiversidad y Biología Experimental y Aplicada (IBBEA - CONICET); Argentina.Fil: Marano, María Rosa. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Marano, María Rosa. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Vojnov, Adrián A. Instituto de Ciencia y Tecnología Dr. César Milstein (ICT – CONICET - Fundación Pablo Cassará); Argentina

Surface barriers of mandarin 'okitsu' leaves make a major contribution to canker disease resistance

This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2014.[EN] Field evaluations have shown that Satsuma mandarin (Citrus unshiu) ‘Okitsu’ is one of the mandarin cultivars that shows substantial resistance to Xanthomonas citri subsp. citri (X. citri), the causal agent of citrus bacterial canker disease. However, the mechanisms underlying this resistance are not well understood. In this study, we have shown that ‘Okitsu’ leaves are nevertheless susceptible to X. citri infection during a period of their development; however, this period is shorter than that seen in the susceptible mandarin ‘Clemenules’ (C. clementina). Under controlled growth conditions, the resistance of ‘Okitsu’ to X. citri was associated with the age of the leaf and was evident in spray-inoculated plants but not in those inoculated by infiltration. Furthermore, X. citri showed reduced attachment and biofilm formation in ‘Okitsu’ leaves compared with ‘Clemenules’. Taken together, our data suggest that structural features of the ‘Okitsu’ leaf surface, such as the physical properties of the cuticle, are involved in the resistance to X. citri.This work was principally supported by the Agencia Nacional de Promocion Cientifica y Tecnologica PICT-2011-1833 to M. R. Marano and by a grant from the Florida Citrus Research and Development Foundation to F. G. Gmitter, Jr. and M. R. Marano. M. A. Chiesa, A. A. Vojnov, A. P. Castagnaro, and M. R. Marano are Career Investigators of CONICET. We thank R Vena for his technical assistance with the confocal microscopy and J. M. Dow and G. Gudesblat for critical review of the manuscript.Favaro, MA.; Micheloud, NG.; Roeschlin, RA.; Chiesa, MA.; Castagnaro, AP.; Vojnov, AV.; Gmitter, FGJ.... (2014). Surface barriers of mandarin 'okitsu' leaves make a major contribution to canker disease resistance. Phytopathology. 194(9):970-976. https://doi.org/10.1094/PHYTO-10-13-0277-RS970976194

Xanthan Induces Plant Susceptibility by Suppressing Callose Deposition

Xanthan is the major exopolysaccharide secreted by Xanthomonas spp. Despite its diverse roles in bacterial pathogenesis of plants, little is known about the real implication of this molecule in Xanthomonas pathogenesis. In this study we show that in contrast to Xanthomonas campestris pv campestris strain 8004 (wild type), the xanthan minus mutant (strain 8397) and the mutant strain 8396, which is producing truncated xanthan, fail to cause disease in both Nicotiana benthamiana and Arabidopsis (Arabidopsis thaliana) plants. In contrast to wild type, 8397 and 8396 strains induce callose deposition in N. benthamiana and Arabidopsis plants. Interestingly, treatment with xanthan but not truncated xanthan, suppresses the accumulation of callose and enhances the susceptibility of both N. benthamiana and Arabidopsis plants to 8397 and 8396 mutant strains. Finally, in concordance, we also show that treatment with an inhibitor of callose deposition previous to infection induces susceptibility to 8397 and 8396 strains. Thus, xanthan suppression effect on callose deposition seems to be important for Xanthomonas infectivity